Amino Acid N-carboxyanhydrides and Polypeptides: Controlled Polymerization, Properties and Biomedical Applications

By: Hua Lu, Ph.D.

Advisor: Prof. Jianjun Cheng, Ph.D.

Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign,

Urbana, IL, 61801, USA

Uniformity of polymeric materials on the molecular level (e.g. chemical composition,

molecular weight (MW), molecular-weight distribution (MWD), and tacticity) determines

their physicochemical properties (e.g. morphology, secondary structure and higher

ordered architecture), and predict their ultimate functions and applications. For instance,

biomacromolecules such as proteins and nucleic acids are evolved with extremely high

uniformity such that all their biological functions are directly or indirectly derived from

and elegantly controlled by their primary structures (chemical compositions and

sequences). Synthetic polymers, on the other hand, are far less uniform compared to those

natural biopolymers and thus represents a major challenge for synthetic polymer chemists.

From a materials science point of view, it is also of great importance to control the

molecular uniformity of synthetic macromolecules in order to achieve desirable

properties and sophisticated functions similar to natural biopolymers. Thanks to the

recent development of living polymerizations methodologies, great improvement have

been made on controlling uniformity of polymeric materials.

Polypeptides are emerging biomaterials receiving increasing attention on various

applications including drug delivery, gene therapy, antimicrobial and tissue engineering.

The ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) is

widely used for preparation of synthetic polypeptides in gram scale. However, controlled

polymerization techniques of NCAs are limited and far from optimized. The primary

goals of my Ph.D. dissertation are to: a) develop controlled/living polymerization

techniques of NCAs for the preparation of high uniform polypeptides; b) study and

understand the chemical and biophysical properties of polypeptides; c) apply novel and

uniformed synthetic polypeptides for biomedical applications.

For the first time, I introduced organosilican amines, namely hexamethyldisilazane

(HMDS)[1] and various N-trimethylsilyl (N-TMS) amines[2], as the initiators for the ROP

of NCAs. This simple and robust system controls the polymerizations remarkably well

(Figure 1). Predicable MWs and narrow MWDs (< 1.2) of homo- and block-

polypeptides can be easily prepared via this technique. Mechanistic study unambiguously

illustrated that the polymerization proceeds through a unique trimethylsilyl carbamate

structure at the chain-propagation center, which revealed an unprecedented mechanism of

NCA polymerization (Figure 1). In addition, the system allows facile end group and

side-chain functionalization[3] of the polypeptides. The introduction of new functional

groups (e.g. alkyne, alkene, azide or nobornene, etc) not only expands the scope of

polypeptides, but also provides extra “chemical handles” facilitating further manipulation

of the structure, properties and functions of the materials (Figure 2). Moreover, I

demonstrated that the one-pot integration of NCA ROP with ring-opening metathesis

polymerization (ROMP) is a powerful and convenient approach for the preparation of

hybrid brush-like polypeptides with complex architectures while maintaining the high

uniformity of both polymerization chemistries (Figure 3a).[4] By using N-TMS amine

functionalized olefin as chain transfer agent, well-defined ROMP-polypeptides block

copolymers also become easily accessible (Figure 3b).[5]

Careful studies on the biophysical properties and self-assembled nanostructures were then

performed. Fascinated about how natural proteins fold into hierarchical conformational

architectures, I focused my investigation on the secondary structure (e.g. α-helix) of the

polypeptides. Using a serial of poly(γ-benzyl-l-glutamate) (PBLG)-grafted brush

polymers, the results indicated that within the special topology of those brush-like

polymers, PBLG, a classical α-helical polypeptide, adopts interrupted helical structure

that can be represented by a “broken rod” model (Figure 4).[6] The helical content of

polypeptides highly relied on the grafting density and length of side-chain PBLGs.

Furthermore, controlled supramolecular polymerization of poly-L-glutamic acid (PLG)-

grafted brush polymers, which resulted in tubular super-helix in macroscopic scale, was

achieved in aqueous solution by taking advantage of the tunable secondary interactions of

PLG (Figure 5).[7] The resulting tubular supramolecular structures, with external

diameters of hundreds of nanometers and lengths of tens of micrometers, are stable and

resemble to some extent biological superstructures assembled from proteins. This study

shows that highly specific intermolecular interactions between macromolecular

monomers can enable the cooperative growth of supramolecular polymers. Most

interestingly, I developed a family of ionic polypeptides with unusual helical stability.[8, 9]

Incorporating charged amino-acid residues to improve peptide solubility, however,

usually leads to reduced helical stability because of increased side-chain charge repulsion,

reduced side-chain hydrophobicity and the disruption of intramolecular hydrogen

bonding. Surprisingly, we discovered that water-soluble, ultra-stable α-helical

polypeptides can be produced by elongating charge-containing amino-acid side chains to

position the charges distally from the polypeptide backbone. The strategy has been

successfully applied to the design and synthesis of water-soluble polypeptides bearing

long, charged side chains and various functional moieties that possess unusual helical

stability against changing environmental conditions, including changes in the pH,

temperature and the presence of denaturing reagents (Figure 6).

I then tested the α-helical cationic polypeptides for nucleic acid delivery applications.

The motivation of the study is to identify efficient transfection reagents and explore how

the helical conformational structure of polypeptides affects the mechanism and efficiency

of transfection. In a screen of a small library of α-helical cationic polypeptides, several

high-efficiency and nontoxic transfection agents that outperform polyethyleneimine, a

well-recognized transfection agent for gene delivery, were successfully identified. Most

significantly, our preliminary data indicate that the helicity of the polypeptides is

essential for their performance, and enhanced membrane disruption is a likely source of

their transfection efficiency (Figure 7).[10] Further investigations on these α-helical

cationic polypeptides for siRNA delivery showed similar results compared to the results

of gene delivery: the enhanced transfection of the top performing α-helical cationic

polypeptide is again linked to its helical conformation and enhanced membrane

disruption ability (manuscript submitted). This discovery was, to my best knowledge, the

first report bridging the conformational structure of materials and their functions in

nucleic acid delivery. The scope and application of my polypeptides was further

demonstrated in anticancer drug-delivery. A recently established delivery system in our

lab exploiting PLG-camptothecin as one of the key components showed promising

efficacy both in vitro and in vivo (Figure 8).[11]

In conclusion, my PhD dissertation traverses a broad range of topics from fundamental

research to materials development in the frontier of chemical science including: living

polymerization methodology, functional polymer synthesis, polymer physics, self-

assembly, and nanomedicine. My research especially emphasized on how controlled

polymerization chemistry and well-defined molecular uniformity impact the properties

and functions of the materials. These works greatly boosted the advance of NCA and

polypeptides field as evidenced by more than 10 peer-reviewed papers published in top-

tier Journals. The research also generated broad and general impact and interests as

demonstrated by several scientific-journal-highlighting, cover featuring, and mass media

disclosure.

References:

[1] H. Lu, J. J. Cheng, J. Am. Chem. Soc. 2007, 129, 14114. [2] H. Lu, J. J. Cheng, J. Am. Chem. Soc. 2008, 130, 12562. [3] H. Lu, Y. G. Bai, J. Wang, N. P. Gabrielson, F. Wang, Y. Lin, J. J. Cheng,

Macromolecules 2011, 44, 6237. [4] H. Lu, J. Wang, Y. Lin, J. J. Cheng, J. Am. Chem. Soc. 2009, 131, 13582. [5] Y. G. Bai, H. Lu, E. Ponnusamy, J. J. Cheng, Chem. Commun. 2011, 47, 10830. [6] J. Wang, H. Lu, Y. Ren, Y. Zhang, M. Morton, J. Cheng, Y. Lin, Macromolecules

2011, 44, 8699. [7] J. Wang, H. Lu, R. Kamat, S. V. Pingali, V. S. Urban, J. J. Cheng, Y. Lin, J. Am.

Chem. Soc. 2011, 133, 12906. [8] H. Lu, J. Wang, Y. G. Bai, J. W. Lang, S. Y. Liu, Y. Lin, J. J. Cheng, Nat.

Commun. 2011, 2, 206. [9] Y. F. Zhang, H. Lu, Y. Lin, J. J. Cheng, Macromolecules 2011, 44, 6641. [10] N. P. Gabrielson, H. Lu, L. Yin, D. Li, F. Wang, J. Cheng, Angew. Chem. Int. Ed.

2011, online early view. [11] K.-J. Chen, L. Tang, M. A. Garcia, H. Wang, H. Lu, W.-Y. Lin, S. Hou, Q. Yin, C.

K. F. Shen, J. Cheng, H.-R. Tseng, Biomaterials 2012, 33, 1162.

Figure 1| HMDS/N-TMS amines mediated Controlled ROP of NCAs. Chemical scheme of HMDS/N-TMS amines initiate controlled NCA polymerizations for the preparation of polypeptides and the plausible mechanism involved in the polymerization.

Figure 2| Side-Chain Functionalization of Polypeptides. Controlled Polymerization of a novel vinyl group functionalized NCA monomer, VB-Glu-NCA, rendered the facile synthesis of side-chain functionalized polypeptides. (Featured as the cover imagine (right panel) of the Macromolecules, Volume 44, Issue16, Augest 23, 2011.)

NHTMS

ROMP

NHTMS

NHTMS

NHTMS

NCA-ROP

ROMP

NHTMS

TMSHN

NCA-ROP

(a)

(b)

TMSHN NHTMS

ROMP monomer

TMSHN NHTMS

chain-transfer agent ROMP polymer polypeptides

Figure 3| Integration of the ROP of NCAs with ROMP for the preparation of hybrid

polypeptides. (a) Synthesis of polypeptide-grafted brush-like copolymers using an N-TMS amine bearing ROMP monomer; (Highlighted by C&EN, issue of Sept. 14, 2009.) (b) synthesis of linear hybrid block copolymers via an N-TMS amine functionalized chain-transfer agent.

Figure 4| “Broken Rods” of polypeptides within brush-like polymers. Cartoon illustration of α-helical conformation of linear PBLG (a) and the interrupted α-helical conformation of PBLGs within a brush-like polymer (b)

Figure 5| Supramolecular Polymerization of polypeptides-grafted brush polymers. (A) Chemical structure of polypeptide-grafted brush-like polymers. (B) pH induced helix-coil transition of polypeptides in brush-like polymers; (C) TEM image of a brush-like polymer, PN9-g-Glu100, after 1 day of incubation at pH 7 at 4 ˚C. Sample was stained by uranyl acetate. (D) Schematic illustration of the supramolecular polymerization of polypeptide-grafted comb polymers into tubular superstructures. (E), (F), (G) and (H) TEM and SEM images of supramolecular structures assembled from PN9-g-Glu100 in solution. (Highlighted by C&EN, issue of Aug. 15, 2011.)

Figure 6| Synthesis and Characterization of ionic helical polypeptides with unusual

stability. (a) Schematic illustration of polypeptide with charged side chains and the postulated helix-coil transition in response to the length of the side chains; (b) Synthesis of PVBLG-X polymers; (c) CD spectra of various polypeptides bearing charged side-chains in aqueous solution at pH 1; (d) The pH dependence of the residue molar ellipticity at 222 nm for (PAHG)57, (PVBLG-1)60, (PVBLG-6)44, (PVBLG-7)40 and (PLL)75; (e) CD spectra of (PVBLG-1)60 in a mixed solvent of MSA and H2O. (Reported by NSF, ScienceDaily, PhysOrg, EurekAlert! and ChemistryViews etc.)

Figure 7| Cationic helical polypeptides for gene delivery. (a) Chemical structures of PVB-L-G-8 and PVB-D,L-G-8; the difference on the configuration of the α–carbons of the two polymers were highlighted; (b) Circular dichroism spectra of helical PVB-L-G-8 and random coil PVB-D,L-G-8 in water; (c) In vitro transfection of COS-7 cells transfected with complexes of PVB-L-G-8 and PVB-D,L-G-8; 25 KDa PEI was included as a control; (d) Calcein internalization study in COS-7 cells treated with PVB-L-G-8 and PVB-D,L-G-8. (e) cartoon illustration of enhanced endosomal release of plasmid gene mediated by cationic helical polymer (highlighted by Chemical and Engineering News, issue of Dec. 19, 2011; Reported by ScienceDaily, PhysOrg, and EurekAlert! etc.)

Figure 8| polypeptides-based drug delivery system for anticancer purpose. (a) Schematic representations of the self-assembly synthetic method for the production of CPT-grafted PGA encapsulated Supramolecular NanoParticles (CPT-PGA SNPs) from the respective molecular building blocks and CPT-PGA (camptothecin-grafted poly(L-glutamic acid)); (b) TEM image of 37-nm CPT-PGA SNPs; (c) In vivo anti-tumor efficacy studies of 37-nm CPT-PGA SNPs (13.6 mg CPT equivalent/kg) along with controls, i.e., free CPT (13.6 mg/kg), PBS and PGA SNPs (equivalent to the amount of SNPs in the CPT-PGA3SNPs group). LLC tumor-bearing C57Bl/6 mice were treated with different groups via intravenous injection at day 1, 6 and 11 (except for CPT group, which was only treated once at day 1 via intraperitoneal injection). 37-nm CPT-PGA SNPs showed delayed tumor growth from day 6 compared to PGA SNPs group and also significantly outperformed CPT group from day 4 (*p < 0.05; **p < 0.01).